Heparin, a glycosaminoglycan, is a highly sulfated polyanionic polysaccharide composed of alternating residues of N-acetyl-glucosamine and glucuronic acid, and is the most widely used clinical anticoagulant. The major clinical applications for heparin include treatment of deep vein thrombosis (DVT) and thromboembolism (TE), prophylactic treatment of patients at high risk for DVT and TE from numerous medical conditions, post-operative prevention of DVT and TE, and prevention of clotting and thrombus formation resulting from interventions in the circulatory system. The circulatory system intervention procedures include cardiovascular diagnostic procedures, catheterization, surgery of the heart and vessels, and many other procedures including extracorporeal circulation, such as hemodialysis, use of artificial organs, and organ transplantation. However, once the procedure has been completed, in many cases the anticoagulation effects of heparin must be neutralized or reversed in order to prevent the patient from bleeding. Protamine is the only agent approved by the FDA for heparin reversal, and administration of protamine has a high incidence of adverse hemodynamic and anaphylactic events. Low molecular weight heparins (LMWH) are being used increasingly as a substitute for conventional unfractionated heparin (UFH) in thromboprophylaxis for numerous medical conditions and for post-surgical prophylaxis, e.g., following hip or knee replacement. Treatment of these patients with either UFH or LMWH results in significant incidence of serious bleeding. Protamine can neutralize the activity of UFH but is not effective in neutralizing LMWH in vivo.
The protamines, purified from fish sperm, are a family of basic proteins rich in arginine residues. Protamine appears to neutralize heparin's biologic effects by overwhelming the carbohydrate with cationic charges. Unfortunately, it may be toxic due to high charge density or distribution of charged residues and its administration frequently causes such problems as hypotension, pulmonary artery hypertension, myocardial depression, complement activation, thrombocytopenia, and leukopenia.
Glycosaminoglycans (GAG)s such as heparin also modulate enzyme activities (e.g., of antithrombin III or heparin cofactor II), regulate cell behavior (e.g., cell adhesion, growth, and differentiation), and control the function of extracellular matrices (e.g., diffusion of ions through basement membranes, and fibrillogenesis and lateral associations of collagens), largely through non-covalent interactions with proteins. (Jackson, R. L., et al., Physiol. Rev. 71:481-539, 1991; Lindahl, U. and M. Hook, Ann. Rev. Biochem. 47:385-417, 1978; San Antonio, et al., Connective Tissue Res., 37:87-103, 1998; WO00/45831). Although many proteins exhibit high affinity interactions with heparan sulfate, heparin, and other GAGs, the specificity of such interactions has been defined for only a small number of them. As heparan sulfates and heparin are among the most structurally diverse and biologically active GAGs, their protein-interactive features have been the most thoroughly studied.
A specific heparin pentasaccharide sequence is known to be an antithrombin III binding site. The site is a pentasaccharide composed of a 6-O-sulfated glucosamine in the first position, a 3-O-sulfated central glucosamine, two N-sulfated glucosamines, and a carboxylated iduronic acid. Other modifications may increase the activity of heparin on antithrombin III, but are not essential for activity.
Cardin and Weintraub identified two potential consensus sequence motifs for heparin-binding, X-B-B-X-B-X or X-B-B-B-X-X-B-X, where X represents a hydropathic or uncharged amino acid, and B a basic amino acid (Cardin, A. D. and H. J. R. Weintraub, Arteriosclerosis 9:21-32, 1989). For example, such consensus sequences were identified in proteins including apolipoprotein B-100, apo E, and vitronectin. Molecular modeling of these consensus sites predicts the arrangement of amino acids into either α-helices or β-strands. This allows for the clustering of noncontiguous basic amino acids on one side of the helix, thus forming a charged domain to which GAGs could bind. Indeed, for some heparin-binding proteins, disruption of the heparin-binding consensus sequences hinders heparin binding. For example, chemical modification of the heparin-binding consensus site in thrombospondin (Lawler, J. and R. O. Hynes, J. Cell Biol. 103:1635-1648, 1986) or site-directed mutagenesis of a heparin-binding sequence in fibronectin (FN) (Barkalow, F. J. B. and J. E. Schwarzbauer, J. Biol. Chem. 266:7812-7818, 1991) eliminates or diminishes heparin-binding affinity. On the other hand, peptide mimetics of proposed heparin binding consensus sequences often fail to reveal the high affinities demonstrated by the native heparin-binding proteins (Conrad, H. E, Heparin-Binding Proteins. Academic Press, 1998). Proteins often contain multiple, distal heparin-binding sequences that may come into proximity upon protein folding or multimerization, hence enabling binding through cooperativity.
The heparin-binding domain of von Willebrand factor resembles the motif XBBXXBBBXXBBX, a palindromic sequence in which the spacing and clustering of basic residues is important for heparin binding (Sobel, M., et al., J. Biol. Chem. 267:8857-8862, 1992). A third novel sequence has been demonstrated to be sufficient for weak heparin-binding in thrombospondin: WSXW (Guo, N. H., et al., J. Biol. Chem., 267:19349-19355, 1992). However, for high affinity binding, this sequence must be flanked by basic residues. Other proteins including type I collagen (Sweeney, S. M., et al., Proc. Natl. Acad. Sci. USA 95:7275-7280, 1998), extracellular-superoxide dismutase (Sandstrom, J., et al., J. Biol. Chem., 267:18205-18209, 1992), and mast cell chymases (Matsumoto, R., et al., J. Biol. Chem., 270:19524-19531, 1995), bind heparin via highly-basic binding regions which do not conform to any consensus sequence. In fact, in certain proteins, domains rich in basic amino acids have sometimes been shown to be unimportant for heparin binding. For example, the two heparin-binding consensus sequences identified in the FGFs were shown not to mediate heparin-binding (Wong, P., et al., J. Biol. Chem., 270:25805-25811, 1995; Thompson, L. D., et al., Biochem., 33:3831-3840, 1994).
GAG structure may also play a role in determining binding affinity and selectivity for proteins. A classic example is the antithrombin-binding site on heparin, which is present on only about one third of heparin chains (Lam, L. H., et al., Biochem. Biophys. Res. Commun., 69:570-577, 1976), but which has a thousand-fold greater affinity for antithrombin III than the overall heparin structure (Lee, M. K., and A. D. Lander, Proc. Natl. Acad. Sci. USA, 88:2768-2772, 1991). Several other sequences or structural motifs have been identified in GAGs which underlie their binding interactions with basic fibroblast growth factor (bFGF) (Maccarana, M., et al., J. Biol. Chem., 268:23898-23905, 1993), lipoprotein lipase (Parthasarathy, N., et al., J. Biol. Chem., 269:22391-22396, 1994), and interleukin-8 (Lindahl, U., et al., J. Biol. Chem., 273:24979-24982, 1998).
Other aspects of GAG structure also contribute to specific interactions with proteins. For example, heparin displays high affinities for sequences in short basic peptides with contiguous clusters of basic amino acids, whereas heparan sulfate displays high affinities for those sequences in which clusters of basic amino acids are separated by non-basic residues (Fromm, J. R., et al., Arch. Biochem. Biophys. 343 (1):92-100, 1997). Such binding preferences may relate to the increased spacing between highly sulfated domains found throughout heparan sulfate as compared with the more uniformly densely sulfated heparin. Heparin is capable of binding to a wide array of proteins, due to its high degree of flexibility.
There is a long felt need in the art for heparin-binding peptides which have greater affinity for heparin and a greater ability to neutralize heparin activity and LMWH activity and little or no hemodynamic toxicity compared to present heparin-binding peptides. The present invention satisfies these needs.